There are various set-ups for measuring the oxygen uptake of any organism. Many of these set ups are boiling tubes or a test tube connected to a scale. Air is then drawn in from the surroundings and the bubble moves towards the invertebrates in the boiling tube. This set up is shown below.
However the problem arises when this set up is used when the invertebrates are put into a water bath. The air inside the boiling tube will heat up and therefore will expand. The air being drawn in from the outside to push the bubble along is at a lower pressure. As a result the air inside the boiling tube will exert a force on the bubble and push it outwards and away from the invertebrates. This will therefore not give an accurate indication of the volume of oxygen produced, as the bubble will not move as far as it should. This can be seen in the diagram below.
Using a closed system device of measuring oxygen consumption can eliminate this problem. This is the case for the manometer, which is connected to a control tube. When the invertebrates are placed in a water bath both the tubes are at the same temperature, and therefore the same pressure. Therefore the manometer fluid will not move away from the invertebrates, as there is an equal force to oppose this increase in pressure. As a result the true oxygen consumption can be measured. For this reason I have decided to use a manometer to measure the rate of oxygen consumption.
The reason for using soda lime is so that any carbon dioxide is absorbed. When the maggots respire they produce carbon dioxide. If soda lime was not present, the manometer fluid in the manometer would not move, as the volume of gas is not changing. When soda lime is placed in the tubes, the carbon dioxide given off by the maggots from respiration will be absorbed. As oxygen is being used up, the volume in the tube will decrease and this will push the manometer fluid towards the tube containing the invertebrates.
Below is a diagram showing how I intend to set up the apparatus.
Variables
There are many variables that affect the rate or respiration, so I must take these into account when I am doing my experiments. These variables are detailed below.
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Temperature- Respiration involves enzymes. Glucose is quite a stable molecule, so has a fairly high activation energy. This must be overcome before any of the glucose can be oxidised, so enzymes are used to lower this energy level. Therefore the maggots must remain in a known temperature so the rate of respiration is steady. As temperature is a variable in my experiment, I must make sure the temperature of the maggots remains constant throughout the experiments. I will vary the temperature from 10 degrees Celsius to 60 degrees Celsius in 10*c intervals.
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Mass of Maggots- Clearly more maggots means more respiration, and therefore more oxygen is consumed. I must therefore keep the mass of the maggots constant.
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Forms of Respiration- There are two forms of respiration- aerobic and anaerobic. In my experiments I am relying on the fact that the maggots will consume air- therefore respiring aerobically, and this will cause the bubble to be pushed towards the maggots. If at times they respire without oxygen- i.e. anaerobic respiration- the rate of oxygen uptake will be affected and will give inaccurate results. I must therefore keep this in mind when carrying out my experiments.
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Concentration of Enzymes/Substrates- Respiration is an enzyme-dependant process and therefore the concentration of enzymes and substrates must be kept constant. This is not something I will be able to control in my experiment, except by keeping the mass of the maggots constant. I must therefore bear this in mind when carrying out my investigation.
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Life Cycle- Just like any other living organism the maggots are part of a life cycle. This cycle is shown below.
Egg
Adult winged Fly Larva (maggot)
Pupa
As the maggot gets older and progresses through the life cycle it becomes a more specialised organism. A fly is a much more complex stage of the organism than the maggot, and so will respire faster. Although it is easy to see the differences between a fly and maggot, I must take into account the age of the maggot increases during the 2 weeks of my investigation. I must therefore also take this into consideration when carrying out my investigation.
Measurements
There are several measurements I must take when carrying out the experiment.
- I will need to record the initial position of the manometer fluid and the distance it has travelled after various time periods. I can then work out the cumulative oxygen consumption.
- The temperature around the maggots
- Mass of soda lime in each boiling tube
- Mass of maggots in the boiling tube
To ensure the experiment is fair I will allow the maggots time to acclimatise to the new environment. I will then take readings for several minutes and then repeat each temperature twice. This will enable me to account for any anomalous results I may acquire during the experiments.
The table below shows the main sources of error and how I have planned to reduce the errors.
Risk Assessment
During my investigation there are various safety issues that I must abide by to ensure my experiment is safe. These are detailed below:
- I will be using a syringe to insert the manometer fluid into the manometer, which can easily cut you and injure someone. Therefore I will make sure that I cover the needle back up when I have finished using it. This will provide a safe working environment.
- Soda Lime is corrosive so I must ensure it does not come into contact with my skin. Therefore I will use a spatula when I need to remove some from the bottle. If I do come into contact with any I will immediately wash my hands thoroughly.
- There is a lot of glassware in my experiment so I will make sure I am careful when using them. If I do drop something I will clear the glass up using a dustpan and brush, whilst making sure I don’t touch any of the shattered pieces of glass.
- Finally I will be using a water bath at temperature up to 60 degrees Celsius. I will therefore have to work carefully and if I do burn myself I will immediately rinse my hand under cold water.
Preliminary Results
Example: To calculate the average cumulative volume of oxygen produced per minute per gram at 2 minutes for room temperature:
Value of Pi x (Radius of Manometer)2 x Distance moved by bubble
Mass of Maggots
Therefore: 3.141592654 x (0.4)2 x 12 = 1.16 mm3 min-1 g-1
5.2
From the preliminary results you can see that as the temperature is raised from room temperature to 35 degrees Celsius, the cumulative volume of oxygen produced over a period of 6 minutes increases- i.e. the rate of respiration increases. This supports the simple prediction I made, in which I stated the rate of respiration would rise which can be clearly seen from the graph. I can now go into more detailed scientific theory of how the rate of respiration has increased.
Theory
Respiration is a process in which organic molecules are broken down in several stages to release chemical potential energy. This is then used to synthesise adenosine triphosphate (ATP). Usually the organic molecule is glucose, but fatty acids and amino acids can also be used if glucose is not present.
The 4 main stages of respiration are glycolysis, the link reaction, the Krebs cycle and oxidative phosphorylation.
Glycolysis is the stage in which glucose is broken up, and this occurs in the cytoplasm of a cell. Initially 2 ATP units are needed to break down the glucose (6-carbon molecule) into 2 molecules of pyruvate (3-carbon molecule). However, during the steps between the splitting of glucose and formation of pyruvate, energy is released and is then used to make 4 ATP units. The diagram below shows the glycolytic pathway.
From the diagram above you can see the steps in between the break down of glucose and the formation of pyruvate. Firstly the glucose, which consists of 6 carbons, is phosphorylated, which is a process requiring energy. As it is phosphorylated twice, to form hexose bisphosphate, 2 units of ATP are used. Glucose is very rich in energy, but is quite unreactive due to having a stable structure. It is therefore broken down to give 2 molecules of triose phosphate. 2 hydrogen atoms are then removed from this molecule by 2 nicotinamide adenine dinucleotide (NAD) molecules to form 2 molecules of pyruvate. The diagram below shows the structure NAD.
Therefore in summary glycolysis results in the net increase of two ATP molecules. However, pyruvate still contains a great deal of chemical potential energy, which is released in the next stages of respiration. The next stage of the process is the Link reaction, which involves many enzymes. These work to lower the activation energy of the reactions and their mechanisms will be discussed later on.
During the Link reaction, pyruvate is decarboxylated- i.e. carbon dioxide is removed. This is very significant in my investigation, as this carbon dioxide is what will be absorbed by the soda lime, and will therefore allow me to calculate the rate of oxygen uptake.
Pyruvate is then dehydrogenated and combined with coenzyme A (CoA) to form acetyl CoA, which is a 2-carbon molecule. Once again NAD is the carrier which removes the hydrogen atoms and forms reduced NAD.
The third stage of the respiration process is known as the Krebs cycle, which is a closed pathway of enzyme-controlled reactions. The product of the Link reaction (Acetyl-CoA) combines with oxaloacetate, a 4-carbon compound, to form citrate, a 6-carbon compound. Like pyruvate, this is then decarboxylated and dehydrogenated in several steps, and eventually oxaloacetate is regenerated to allow the cycle to start again.
Each turn of the cycle results in 2 carbon dioxide molecules forming, one FAD molecule and 3 NAD molecules are reduced and one ATP molecule is generated. The diagram below shows the Link reaction and the Krebs cycle.
It is in the final stage of respiration that the bulk of ATP is produced, and it is this stage which requires oxygen. It is in this stage that the hydrogen atoms being carried by the reduced NAD and reduced FAD are put to use, and the stage is known as Oxidative Phosphorylation.
During glycolysis, the Link reaction and the Krebs Cycle only a few molecules of ATP have been produced. It is during oxidative phosphorylation that most of the energy locked in the original glucose molecule will be released. The electron transport chain is a network of electron-carrying proteins located in the inner membrane of the mitochondrion. Reduced NAD and reduced FAD pass to the electron transport chain, and here hydrogen atoms are released from the two carriers. In doing so one ATP molecule can be synthesised. The hydrogen atoms then break down into hydrogen ions (H+) and electrons. The hydrogen ions remain in the mitochondrial matrix, whilst the electrons are transferred to the first electron carrier. As it passes between the three electron carriers, the energy level of the carriers, in relation to oxygen, decrease. This releases energy, which is used to synthesise ATP. In this way two more molecules of ATP are produced. Finally the electron reaches the final electron acceptor, which is also located in the mitochondrial matrix, and is oxygen. 2 hydrogen ions will also be drawn up and the oxygen is reduced to water.
This is why in my investigation the manometer fluid in the manometer will move towards the maggots. The maggots require oxygen for this last stage of respiration, which is what I am measuring to calculate the rate of respiration.
However this only accounts for 3 of the 28 molecules of ATP formed during oxidative phosphorylation. The other ATP molecules are synthesised due to the process of chemiosmosis. The energy released by the electron transport chain is also used to pump hydrogen ions into the mitochondrial intermembrane space. Due to this the concentration of hydrogen ions in the intermembrane space increases. This sets up a concentration gradient, and the hydrogen ions then pass back through the membrane into the mitochondrial matrix via protein channels. In each channel is ATP synthase, which acts as an enzyme and uses the potential energy of 3 hydrogen ions to convert ADP and Pi (inorganic phosphates) to ATP. The diagrams below show the process of oxidative phosphorylation and also how ATP synthase works. ATP synthase is an enzyme, which shows that respiration is enzyme-dependant and so I must also consider how enzymes work to make this process possible.
Now that we have seen how respiration is able to release energy from glucose, we can consider the role enzymes play in the process. The enthalpy diagram below shows the overall change of glucose into carbon dioxide and water.
Although there are many compounds formed between glucose and the end product, essentially they can all be regarded as one. This is because for each individual step to occur the activation energy must be overcome. The enzymes work by lowering this energy, which allows the glucose to be converted to pyruvate during glycolysis, the pyruvate to be converted to Acetyl-CoA during the Link reaction etc. I will now go into more detail about how the many enzymes involved in respiration are able to do this.
An enzyme can be defined as a biological catalyst and is affected by the environment it is in. The enzyme has a specific 3-dimensional shape and this means a certain enzyme can break down only substrates with a certain shape.
E.g. For this reason amylase can only break down starch, due to the substrates fitting into the active site. It cannot however break down lipids due the lipid substrates having a structure that does not allow it to fit in the active site of the amylase enzyme.
As stated earlier enzymes are complex 3-dimensional globular proteins. The active site, which is usually a cleft in structure, contains some amino acids that carry out the breakdown of a substance.
H R O
Diagram showing structure of a simple
N C C amino acid
H OH
The enzyme is in the tertiary structure of a protein. It is held together by several bonds, which are hydrogen bonds, ionic bonds, disulphide bonds and hydrophobic interactions between non- polar side chains. The long amino acid chains coiling up on themselves bring about this structure. Hydrogen bonds then form between the –CO groups of one amino acid and the –NH group of another, which hold this shape in place. This is called an α- helix and is the secondary structure. This structure may coil up into a precise three- dimensional shape, which is the tertiary structure. The diagram below shows how hydrogen bonding can form.
The R groups determine the shape of the active site in an enzyme. The large variety of different R groups means different shape active sites can exist, explaining why the enzymes are specific to one substrate type.
The diagram below taken from ‘Biology 1’ illustrates how an enzyme works.
Diagram showing how an enzyme speeds up the breakdown of a substrate
The above diagram relates to how the enzymes in respiration work. In the left diagram we can see that the enzyme and substrate are in a mixture. The substrate moves into the active site of the enzyme. The two then bind and form an enzyme-substrate complex. It is held in place using temporary bonds that form between the R groups of the enzyme’s amino acid and the substrate. These bonds are weak and thus are not covalent.
The lock and key diagram can be used to understand the specific shape of the enzyme. The substrate above fits the shape of the active site, so can bind with it. Any other shape will not fit this active site. A lock and a key theory can resemble this in that if the key, i.e. the substrate, is not the right shape it will not fit in the lock, which is the enzyme.
Finally the interactions between the substrate and active site of the enzyme cause the substrate to break down. The temporary bonds, which form during this process, cause a higher tendency for the breakdown of a substance, which in turn reduce the activation energy. This will be explained in more detail in the next section.
Now that we know what an enzyme is we can see how altering the temperature would cause a change in the rate of the reaction of respiration.
For a reaction to occur the particles must collide with a certain minimum kinetic energy. The size of this kinetic energy needed varies between reactions due to different bond enthalpies. An enzyme works by reducing the activation energy as shown in the diagram below.
Energy
Progress of Reaction
As you can see, the enzyme lowers the activation energy, which gives a greater number of particles the minimum energy required for the reaction to occur. The energy profile diagram shows the smaller peaks, which arise as a result of the enzyme. The first is from the formation of the enzyme-substrate complex. After this stage the enzyme-product complex forms, which also requires energy but less so than the enzyme-substrate complex. Finally the enzyme and products move away, and the enzyme can then be used again.
The molecules during the respiration process have a range of different kinetic energies. Most of the particles will be moving at moderate speeds, others will have slightly greater kinetic energy and some will have slightly less. When the temperature of the reactants rises, they move around faster and have a greater amount of kinetic energy.
This means that of those substrates that collide with the enzymes, the amount of energy of the impact is more likely to exceed the activation energy. The enzyme lowers the activation energy further, so that a larger number of molecules have the required energy and can cause a reaction. This is illustrated in the diagram below.
Number of collisions
With Kinetic Energy,
E
Kinetic Energy (E)
The above diagram shows that only a small proportion of the molecules have the energy E to overcome the activation energy (which in this case is 50kg mol-1), and to cause a reaction to occur. If we now however raise the temperature the graph will look like the one shown below.
Distribution curves showing effect of a temperature rise of 10K on the proportion of reactions with greater than 50kg mol-1
From the graph you can see that by increasing the temperature by 10 Kelvins the graph has shifted to the right- i.e. there is a higher average kinetic energy of each particle. There is a much higher proportion of molecules with greater than 50kg mol-1, which means more collisions will be successful enough for a reaction to occur.
Therefore increasing the temperature will raise the average kinetic energy of the reactants, which will enable a larger number of reactions to occur. This is why think raising the temperature will increase the rate of respiration. However raising the temperature too far will cause the enzyme to become denatured. This means the bonds holding the tertiary structure of the enzyme together will be overcome, and the active site will have altered in shape. As a result the temporary bonds that occur between the substrate and the enzyme cannot form. This would therefore cause the enzyme to become of no use so the reaction progress will decrease and eventually stop.
Rate of Reaction
Temperature
The diagram above summarises how the overall reaction rate varies with temperature. At a low temperature the substrate and enzyme have less kinetic energy so their collisions are weak and generally insufficient to overcome the activation energy. When the temperature is raised, however, their kinetic energy increases so there is a higher likelihood the enzyme-substrate complex can be formed. The rate will then eventually peak, which is known as the optimum rate. The rate will then begin to decrease when the temperature is too high. This is because many of the bonds holding the tertiary structure together will break (e.g. hydrogen bonds and ionic being weak will break but disulphide bridges which are strong covalent bonds may not break), and more and more enzymes will become denatured. The diagram below shows a rough representation of how the structure of the enzyme, in particular the active site, changes when it becomes denatured and why it is not able to work.
Diagram taken from Biology 1 Advanced Sciences page 42 by Mary Jones, Richard Fosbery and Dennis Taylor
Diagram taken from Salters Advanced Chemistry: Chemical Ideas 10.2 page 225
